Rab11A Controls the Biogenesis of Birbeck Granules by Regulating Langerin Recycling and Stability -- Uzan-Gafsou et al. 18 (8): 3169 Data Supplement - Supplemental Material -- Molecular Biology of the Cell

Rab11A Controls the Biogenesis of Birbeck Granules by Regulating Langerin Recycling and Stability
Mol. Biol. Cell Uzan-Gafsou et al. 18: 3169

Supplemental Material

This article contains the following supporting material:

Figure S1 - Invertase is a ligand for Langerin in M10-22E cells. After endocytosis of Cy3-invertase (10 μg/ml) for 1 hour at 37°C, M10-22E cells (A-D, left panels) were incubated with a rabbit anti-Langerin serum recognizing an epitope in the cytoplasmic domain (A, middle panel), a rabbit anti-Rab11 serum (B, middle panel), an anti-TfR mAb (C, middle panel) or the mAb 6C4 (D, middle panel). The cells were then incubated with donkey anti-rabbit or anti-mouse A488 antibodies. Higher magnification inserts are included to improve visualization of the colocalization of invertase with the various markers in merged images (A-D, right panels). Scale bars correspond to 10 μm.
Figure S2 - Cells were infected with recombinant adenoviruses encoding the GDP-bound dominant negative mutant GFP-Rab4S22N (A) or the GTP-bound chimeric Rab protein GFP-Rab4Q67L (B). 18 to 20 hours after infection, the cells were labeled with the anti-Langerin mAb DCGM4 (A-B), which was revealed with donkey anti-mouse Cy3. Scale bars correspond to 10 μm.
M10-22E cells infected with GFP-Rab4S22N (C) or GFP-Rab4Q67L (D) adenoviruses were allowed to internalize Cy3-transferrin (Tf) for one hour at 37°C. Cells infected with Rab4S22N (E) or Rab4Q67L (F) were also incubated with Cy3-invertase (E, F, left panels) for 1 hour at 37°C and then labeled with DCGM4 (E, F, middle panels). Note that internalized invertase masks the epitope of DCGM4 on Langerin in cells overexpressing either Rab4 mutant. GFP staining is shown for the Rab4S22N mutant (E, right panel) and the Rab4Q67L mutant (F, right panel). Merged images (G, H) show the colocalization of internalized Cy3-invertase and Langerin as revealed with an antiserum against the cytoplasmic domain of Langerin and donkey anti-rabbit A488, in cells infected with Rab4S22N (G) or Rab4Q67L (H) adenoviruses. Scale bars correspond to 10 μm.
Figure S3 - Effects of Rab4Q67L recombinant adenoviruses on the morphology of the ERC of M10-22E cells. (A) Uninfected M10-22E cells (Ctrl) were incubated with HRP (10 mg/ml) and/or a gold-labeled anti-Langerin mAb for 30 minutes. BGs (arrows) appear to be located close to the nucleus (Nu) and are continuous, on the left hand side of the figure, with a vacuolar endosomal structure (star) and on the right hand side with a tubular membrane structure (B-D). M10-22E cells were infected for 18 hours with GFP-Rab4Q67L recombinant adenoviruses, in the presence of HRP (10 mg/ml) and/or a gold-labeled anti-Langerin mAb for the last 30 minutes. Cytosolic BGs (arrows) appear to be continuous with and/or in the close vicinity of "multivesicular" endosomes which are located close to the nucleus (B) or the cell surface (C). These endosomes (D) and the BGs (C) are labeled with the 6 nm gold-conjugated anti-Langerin mAb.